Blood glucose remains within a narrow range, primarily due to insulin and glucagon, which are secreted from the beta and alpha cells of the islets of Langerhans, respectively. Although insulin secretion has been well studied the signals that regulate glucagon secretion are still hotly debated. Hypoglycemia is a significant cause of death amongst Type I diabetics and the major limitation upon insulin treatment of hyperglycemia. Thus, it is crucial to understand how alpha cells sense and respond to circulating glucose levels. It is unclear whether glucose is detected directly or whether the alpha cell is under the control of neighboring beta cells, most likely through insulin secretion. This study will develop 2 methods by which we can separate primary rat alpha cells from the effects of neighboring beta cells. The first approach is to obtain pure alpha cell populations using green fluorescent protein (GFP) expression restricted to the alpha cells to tag these cells for purification by fluorescence activated cell sorting (FACS). Techniques will then be developed to optimize the conditions for culturing these purified alpha cells. The use of cell aggregation and cell monolayers will be examined to determine which method gives the tighter regulation of glucagon secretion. The other method is to down-regulate insulin receptor expression in the alpha cells. The aim of the second aspect of this study is to identify steps of proglucagon gene expression (RNA levels, RNA stability and proglucagon translation) that are regulated in response to glucose and/or insulin. The identification of such steps would provide novel read-outs by which to study alpha cell responses to nutrients. Furthermore, the regulation of gene expression at mRNA stability and translation is almost always via RNA-protein interactions, the identification of which would provide valuable tools in the dissection of nutrient effects upon the alpha cell. The research described in this proposal will provide new techniques and new read-outs by which the study of the alpha cell responses to its nutrient environment can be studied.